Our long-term goal is to find out how axons in the vertebrate brain determine when and where to form synapses. We focus on laminar specificity, a fundamental determinant of connectivity throughout the brain, whereby neuronal processes confine their arbors and synapses to specific laminae within a target area. Our object of study is the retinal ganglion cell (RGC), because it is relatively accessible, has a well-defined function, and displays exquisite laminar specificity: axons of distinct RGC subsets synapse in specific sublaminae of the optic tectum's retinorecipient lamina (RRL), and their dendrites arborize in specific sublaminae of the inner plexiform layer (IPL), where they receive inputs from lamina-specified subsets of amacrine and bipolar cells. In work supported by this grant, we have identified several molecules likely to regulate laminar selectivity in retina, and now propose to investigate the mechanisms by which they act: (i) We found that four related immunoglobulin superfamily (IgSF) adhesion molecules, Sidekicks-1, Sidekick-2, Dscam and DscamL are critical determinants of an IgSF code that underlies some aspects of sublaminar specificity in the IPL. We will now assess their mechanisms of action and use transgenic methods to mark and manipulate the cells in which they are expressed. (ii) In a search for additional IgSF molecules in retina, we found that JAM-B is expressed by a subset of RGCs, generated mice in which these cells are indelibly labeled, and showed that JAM-B marks a unique population of lamina-specified cells sensitive to upward motion. We will now follow the development of this population, and ask whether JAM-B helps determine their structure or function. (iii) A family of ~60 protocadherins, has been proposed to mediate synaptic specificity in the retina and elsewhere. We previously demonstrated synaptic defects in mice lacking all 22 genes of the gamma protocadherin cluster, but analysis was complicated by neonatal lethality. We have now obtained conditional mutants that are healthy but have defects in retina, so we can directly assess its role. Finally, we will return to studies of laminar targeting in tectum, to determine the relationship between lamina-specific choices made by dendrites and axons of single cells. We will assess roles of Type II cadherins in tectum, and use a novel multicolor transgenic strategy to document laminar patterns of RGC axonal arbors in mouse superior colliculus. Using these strategies we hope to elucidate mechanisms that promote lamina-specific synapse formation and, by extension, synaptic specificity generally. NARRATIVE During development, neurons connect with each other in very specific ways, forming the complex circuits that underlie our mental activities. Our long-term goal is to find some of the cells and molecules that determine when and where these connections, called synapses, form. We are focusing on a particular process called laminar specificity, whereby neuronal processes confine their arbors and synapses to specific slab-like laminae within a target area. Our object of study is the retinal ganglion cell (RGC), the neuronal type that carries visual information from the eye to the brain: it is relatively accessible, has a well-defined function, and displays exquisite laminar specificity both in terms of receiving synapses in the retina and making them in a brain area called the optic tectum or superior colliculus. In work supported by this grant, we have identified several molecules likely to regulate laminar selectivity in retina, and now propose to investigate the ways in which they act. They include a set of four related synaptic proteins called Sidekick-1, Sidekick-2, Dscam and DscamL;a distant relative called JAM-B;and a remarkable family of ~60 closely related genes called protocadherins. In addition, we will study laminar targeting in tectum, so we can determine the relationship between lamina-specific choices made by dendrites and axons of single cells. Using these strategies we hope eventually to elucidate mechanisms that promote lamina- specific synapse formation and, by extension, defects in specificity that may underlie behavioral disorders.